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PubMed1996

[Modulation of cisplatin cytotoxicity by amphotericin B in six human cell lines of medullary thyroid cancer].

Medullary thyroid carcinoma (MTC) is frequently resistant to chemotherapy. In this work, we have studied the effect of cisdiamminedichloroplatinum (II) (CDDP) in six MTC human cell lines and we have tried to reverse the resistance to CDDP with amphotericin B (AmB). We also studied the metabolism of glutathione (GSH) and the presence of the glutathione-sulfotransferase pi (GST pi) mRNA in the MTC cell lines. The cisplatin-induced cytotoxicity was evaluated with the 3-4,5 dimethylthiazol-2,5 diphenyl tetrazolium bromide (MTT) test, the neutral red (NR) uptake and with total GSH measurement in six cell lines, TT cell line and five cell lines that we isolated. The cultures were performed with or without AmB (5 micrograms/mL). Intracellular GSH was measured in TMC cells and compared to the levels obtained in six normal thyroid tissues. The expression of GST pi mRNA was evaluated by Northern blotting in the different cell lines. A CDDP-induced cytotoxicity was obtained in the six cell lines at doses inhibiting 50% of the cellular proliferation (IC50) varying from 6 to 40 micrograms according to the tests and the cells tested. A low concentration of AmB (5 micrograms/mL) potentiated the cisplatin toxicity after a 48-h coincubation of TMC in all cases. GSH levels in TMC cell lines were identical to those found in normal cells. GST pi mRNA was detected in all the TMC lines, except in TT cell line. In conclusion, CDDP was toxic for all the TMC cell lines and AmB potentiated this antitumoral effect. On the contrary, GSH and GST pi do not seem to be involved in the mechanisms of the resistance in these cell lines.

Amphotericin B↗
PubMed1996

The human retinoblastoma gene product suppresses ceramide-induced apoptosis in human bladder tumor cells.

The retinoblastoma gene product, Rb, has previously been implicated as an obligatory component in the antiproliferative effects mediated by the lipid second messenger, ceramide. We have evaluated both the apoptotic effects and the effects on cell cycle distribution of the exogenous cell-permeable ceramide, N-hexanoyl-D-sphingosine, in an Rb-null human bladder tumor cell line, 5637, as well as in retrovirally infected, Rb(+) clones derived therefrom. These cell lines demonstrated comparable sensitivity to N-hexanoyl-D-sphingosine in a neutral red dye uptake assay. Exposure of the Rb-null parental cell line to 20 microM N-hexanoyl-D-sphingosine for 24 h resulted in a classical pattern of DNA fragmentation that was accompanied by apoptotic nuclear morphological alterations. In contrast, the Rb(+) clones demonstrated suppression of DNA fragmentation in response to N-hexanoyl-D-sphingosine. Similarly, the frequency and degree of alteration of nuclear morphology in Rb(+) cells was also suppressed. Flow cytometric analysis of the parental and infected clones indicated that expression of Rb was without effect on their cell cycle distribution, with or without exposure to N-hexanoyl-D-sphingosine for 25 h; tunel assay confirmed that in this time frame apoptotic cells were far less frequent in the Rb(+) clones than in the parental 5637 cells. Human tumor cell lines derived from three other histological origins, breast and prostatic carcinomas and osteogenic sarcoma, also demonstrated very similar cytotoxic sensitivities to N-hexanoyl-D-sphingosine, irrespective of the expression of Rb. We conclude that Rb is not required for ceramide-induced apoptosis and that Rb can actually inhibit the DNA fragmentation and nuclear morphological changes associated with classical apoptosis.

Apoptosis↗
PubMed1996

Functional upregulation of granulocytes labeled with technetium-99m-HMPAO and indium-111-oxinate.

UNLABELLED: Indium-111-oxinate-labeled granulocytes have been used in vivo for several years for the detection of abscesses. Technetium-99-m-hexamethylpropyleneamine oxime (99mTc-HMPAO) labeling has more recently been described. METHODS: The influence of radiolabeling by both radiotracers on adhesion glycoprotein CD11b quantification was studied in quiescent and formyl-methionylleucylphenylalanine (fMLP)-activated neutrophils (PMN). Adhesion was assessed on human umbilical endothelial cells (HUVEC) as well as the repercussion of the granulocyte labeling on HUVEC viability (neutral red) and metabolic activity (MTT). Chemotaxis of PMN was evaluated by measuring migration under agarose with fMLP as chemoattractant. We also measured phagocytosis and the production of hydrogen peroxide induced by staphylococcus aureus. RESULTS: Whereas whole functional integrity is maintained after labeling, most of the functions (CD11b expression, adhesion, HUVEC metabolic activity) are up-regulated while chemotaxis is decreased in the presence of both radiotracers. Indium-111-oxinate induces larger alterations than 99mTc-HMPAO. CONCLUSION: These data were obtained in normal volunteers. In patients, alterations due to the in vitro labeling procedure, in addition to potential functional alterations caused by the underlying pathology, should be taken into account during image interpretation.

Cells, Cultured↗
PubMed1996

[A new osteoblast cell culture system for standardized testing of biomaterials].

For in vitro testing of new biomaterials cultured fibroblasts are employed. In the case of the agar diffusion test survival of cells is involved in the presence of the material to be tested. Further statements on the biological effects of a biomaterial require the use of cell cultures adapted to the tissue concerned and the underlying problem being investigated. In the present study, an osteoblast cell culture system with which implant surfaces in contact with bone can be tested as required by the relevant standards is described. Test bodies made of titanium, polystyrene or copper were used in the conventional agar diffusion test, and were also overgrown with fibroblasts or a cell line of foetal human osteoblasts. For the agar diffusion test, the test criterion was the extent of the inhibition area on staining with neutral red, while for the overgrowth, the mean cell diameter and the number of cells was employed. The phenotype of the osteoblast cell line was determined immunohistochemically by means of alkaline phosphatase or immunohistologically by means of collagen I and osteocalcin. Calcification was demonstrated using the v. Kossa stain. In the case of the osteoblasts, a differentiation of a collagen I and alkaline phosphatase-positive phenotype over an osteocalcin-positive phenotype to an increase in calcium deposition was shown. As in the case of the agar diffusion test, direct overgrowth also revealed no cytotoxic effect for titanium and polystyrene. In contrast, a cytotoxic effect consisting in a decrease in the number of cells and also a left shift in the size distribution was observed for copper. The standard deviations of the individual tests were less for overgrowth than for the agar diffusion test. The culture system for osteoblast cells thus meets the criteria of the EN/DIN 30993-5 in terms of the quality and accuracy of the results obtained. In addition to excluding direct cytotoxicity, this test system offers a new possibility of examining the influence of the material on cell growth. Consequently, it permits a repeatable examination of proliferation and differentiation of the osteoblasts on each material surface.

Animals↗
PubMedDate not supplied

[Effects of electromagnetic radiation of various modes on heart activity (in experiments)].

On spinal cord frogs and isolated interauricle to a partition of heart in vivo and in vitro influence the MICROWAVES of a radiation in continuous and modulated modes on function of heart (9.3 Hz is investigated; 0.348-0.16 and 0.016 mV/sm2, modulation from 1 up to 100 Hz). A possibility of influence of an electromagnetic exposure on heart frequency and rhythm is revealed. Pointing of heart in vitro by a neutral red resulted in large number of % of experience in a stop of irradiated heart.

Animals↗
PubMed1997

Partial characterization of cytoplasmic compartments involved in the endocytic process of Tritrichomonas foetus.

The endocytic pathway of Tritrichomonas foetus, a parasitic protozoan of cattle, was studied using (a) vital dyes, such as Lucifer yellow, neutral red and acridine orange, (b) cationized ferritin, (c) gold-labeled lactoferrin and lectins: HPA, UEA, PNA and LPA, and (d) DAMP (3-(2,4-dinitroanilino) 3' amino-N-methyldipropylamine). Light and confocal laser microscopy as well as transmission electron microscopy were used in this study. Assays were monitored by fluorescence and electron microscopy after exposing the parasites to different conditions. Cells that were incubated at 15 degrees C or 20 degrees C with gold-labeled lactoferrin and processed for electron microscopy show that of 15 degrees C this ligand is found only in an early endosomal compartment and at 20 degrees C it is found in late endosomes but not in lysosomes. Immunocytochemical data from cryosections using DAMP as a pH probe show that T. foetus has acidic compartments, with a pH range of 5.2 to 6.6, with variable morphology, localization and size. Lectin-binding sites and anionic sites were also internalized and appear to be associated with membranes lining the vacuoles. Images of patching and shedding of these sites were also observed when HPA and UEA were used.

Animals↗
PubMed1997

Isolation and morphofunctional characterization of mussel digestive gland cells in vitro.

The aim of the present study was to isolate and characterize digestive gland cells of the bivalve mollusc Mytilus galloprovincialis Lmk. The digestive gland of bivalves is a complex organ composed of digestive and connective tissues but it is also invaded by the reproductive tissue as gametogenesis proceeds. The digestive tissue is comprised of stomach and intestinal epithelial cells, ciliated and non-ciliated duct cells, digestive cells and basophilic cells. the last two cell types are found lining the epithelium of the blind-ending digestive tubules and are the main focus of this study. Two different approaches were assayed for cell isolation, i.e., explant culture techniques and mechanical plus enzymatical digestion techniques. Cell viability was tested by trypan blue exclusion, neutral red uptake and ultrastructural analysis. The explant cultures were often contaminated with bacteria and spermatozoa and, moreover, cells migrating out of the explants possibly corresponded to hemocytes and not to digestive tissue cells. Mechanical plus enzymatical digestion with collagenase was concluded to be the method of choice for digestive cell isolation, with a percentage of about 30% of isolated cells corresponding to digestive cells. The ultrastructure of digestive cells isolated with the latter procedure closely resembled that of mussel digestive cells in vivo.

Animals↗
PubMed1997

A tumor suppressor gene, Cx26, also mediates the bystander effect in HeLa cells.

The connexin 26 (Cx26) gene suppresses the growth of HeLa cells in vitro and in vivo. We explored the possibility that the Cx26 gene not only suppresses growth but can also mediate the bystander effect that is observed in some gene therapy. In gene therapy mediated by the herpes simplex virus thymidine kinase, the toxicity of ganciclovir affects not only the cells transduced with the gene but also affects neighboring tumor cells; it has been suggested that gap junctional intercellular communication (GJIC) may play a role in such a bystander effect. HeLa cells expressing the Cx26 gene (Cx26+) or not expressing the Cx26 gene were transfected with the herpes simplex virus thymidine kinase (tk+) gene, producing Cx26(-)-tk-, Cx26(-)-tk+, Cx26+-tk-, and Cx26+-tk+ cells. By making different kinds of cocultures of these cells, we observed a clear bystander killing effect, assessed by the neutral red toxicity test, in the coculture of Cx26+-tk-/Cx26+-tk+ cells. The bystander effect was markedly prevented by a long-term inhibitor of GJIC, 18-alpha-glycyrrhetinic acid, demonstrating that a major part of the bystander effect seen occurred through Cx-mediated GJIC. These data suggest the possibility of using of Cxs as both tumor suppressor genes and as diffusers of ganciclovir toxicity in therapeutic approaches.

Connexin 26↗
PubMedDate not supplied

Reduced glucose degradation products in bicarbonate/lactate-buffered peritoneal dialysis solutions produced in two-chambered bags.

OBJECTIVES: The aims of the current study were: (1) to determine the effects of peritoneal dialysis (PD) solutions at different glucose concentrations on the growth of cultured cells; (2) to determine whether a bicarbonate/lactate-based solution, as a result of the configuration of its components during heat sterilization in a two-chambered bag, was lower in glucose degradation products than a corresponding lactate-based PD solution; and (3) to determine whether lower glucose degradation corresponded to a decreased inhibition of cell growth. DESIGN: Growth inhibition of cells exposed to lactate-based PD solutions at three different glucose concentrations was determined. Bicarbonate/lactate-based and lactate-based solutions at high glucose concentration (3.86%) were further analyzed for presence of glucose degradation products and inhibition of cell growth. METHODS: Cell growth was determined by neutral red uptake, measured by optical density at 540 nm. Glucose degradation to acetaldehyde or fructose was determined by gas chromatography-mass spectroscopy and high-performance liquid chromatography. RESULTS: Only 3.86% glucose lactate-based PD solution caused significant inhibition of cell growth (p < 0.05). The heat-sterilized, bicarbonate/dlactate-based solution (3.86% glucose) had lower levels of fructose and acetaldehyde than a conventional heat-sterilized, lactate-based solution with the same glucose concentration. Growth of cultured cells exposed to the bicarbonate/lactate-based solution was significantly improved (p < 0.05) over growth in the conventional solution. CONCLUSIONS: The bicarbonate/lactate-based solutions, manufactured and heat-sterilized in two-chambered bags, were lower in glucose degradation products than that corresponding lactate-based PD solutions, and demonstrated improved in vitro biocompatibility as measured by the growth of cultured cells.

Animals↗
PubMed1998

PUVA-induced cell mortality in NCTC 2544 keratinocytes: is it related to the microenvironmental properties of the excited states of psoralens?

The phototoxic effect of psoralen (PSO), 5-methoxypsoralen (5MOP), 8-methoxypsoralen (8MOP) and 4,5',8-trimetylpsoralen (TMP) has been compared on the NCTC 2544 keratinocyte cell line in terms of cell mortality and lipid peroxidation. The order of effectiveness for cell photokilling is TMP, 5MOP >> 8MOP, PSO, whereas a little lipid peroxidation is observed for the four psoralens under study. Oxygen-independent membrane damage seem to play a key role in the lethal photodamage because the biological effectiveness of the most hydrophobic lipid-soluble psoralens, TMP and 5MOP, is about an order of magnitude higher than that of the more water-soluble 8MOP and PSO. In relation to this hypothesis, and in contrast to 8MOP, TMP is readily extracted from cells by ethyl acetate, a good membrane solvent, as shown by GC/MS analysis on cell extracts. The results are discussed in terms of the highly microenvironment-dependent photophysical properties of psoralens. By the measure of the intracellular psoralen concentration, the neutral red uptake and the lipid peroxidation products, this work provides evidence that PUVA therapy-mediated cell mortality is a lipid peroxidation-independent phenomenon.

Cell Line↗
PubMed1998

Projections from dorsomedial part of the subnucleus oralis to the mesencephalic trigeminal neurons innervating the masseter muscle--a PHA-L and HRP double labeling study in the rat.

When PHA-L was injected to dorsomedial part of the subnucleus oralis of the spinal trigeminal nucleus (Vodm), anterogradely labeled varicosities and boutons were observed in the mesencephalic trigeminal nucleus (Vme). The PHA-L labeling was seen mainly in the Vine ipsilateral to the injection side. If the injection sites were placed more medially containing both Vodm and parvocellular reticular nucleus (PCRt), more PHA-L labeling in contralateral Vme was observed. It appeared that the Vodm neurons projected predominantly to the ipsilateral Vme, while neurons in the PCRt adjacent to the Vodm projected bilaterally to the Vme. It was also seen that many PHA-L labeled varicosities and boutons surrounded or contacted on the Vme neurons counterstained with cresyl violet. These contacts were seen frequently in the ipsilateral Vme, occasionally in the contralateral nucleus. Some Vme neurons ipsilateral to the injection side were retrogradely labeled in a solidly-filled appearance following injection of PHA-L into the Vodm. When PHA-L was applied to the Vodm and HRP was injected to the masseter nerve on the same side, the relationship between PHA-L labeled terminals and HRP labeled Vme neurons was studied. The contacts between PHA-L labeled boutons and HRP labeled somata or processes of the Vme neurons were observed occasionally. PHA-L labeled terminals sometimes contacted on none-HRP-labeled somata counterstained with neutral red. Besides the PHA-L labeling in the Vme, the labeling was also seen in the medial parabrachial nucleus ipsilaterally, in the motor trigeminal nucleus bilaterally with ipsilateral predominance. In addition, an outstanding labeling band along the medial border of the principal sensory trigeminal nucleus (Vp) was observed following injection of PHA-L into the Vodm. The labeled terminals were concentrated in dorsomedial part of the Vp (Vpdm) and in juxtatrigeminal nucleus (JX), meanwhile many labeled fibres were distributed between the Vpdm and JX in the intertrigeminal region.

Animals↗
PubMed1998

Dye penetration at a cobalt-chromium alloy--composite resin interface.

This study examined microleakage along the interface of resin systems bonded to cobalt-chromium alloy; a microfine resin with adhesive properties based upon methacrylic acid, and a hybrid resin containing an adhesive phosphate monomer. Specimens were thermocycled between 4 degrees, 37 degrees and 60 degrees C over 1000-10,000 cycles and placed in 0.1% neutral red dye for 7 days. Each was cut diagonally and the interfaces photographed using an optical microscope. Dye penetration was expressed as a percentage of the total length of each edge. The hybrid resin showed significantly less microleakage than the microfine resin.

Chromium Alloys↗
PubMed1998

Stress-induced fractal rearrangement of the endothelial cell cytoskeleton causes apoptosis.

BACKGROUND: Apoptosis, a mechanism of cell death prominent in critical illnesses including disseminated inflammation and multiorgan dysfunction syndrome, is characterized by morphologic changes including cell shrinkage, condensation of organelles, blebbing, and chromatin fragmentation. These phenomena suggest substantial changes in cytoskeletal structure. We hypothesized that stress-induced apoptosis in endothelial cells is, in part, a consequence of a critical cytoskeletal rearrangement. METHODS: Porcine aortic endothelial cells in culture, surrogates for the microvasculature in vivo, were exposed sequentially to Escherichia coli endotoxin (25 micrograms/mL; 18 hours) to induce the inflammatory response and then to sodium arsenite (160 mumol/L; 120 minutes) to induce the heat-shock response, a well-characterized model of stress-induced apoptosis. Laser confocal micrographs of fluorescein isothio-cyanate-labeled phalloidin-stained cells were analyzed to calculate the border fractal dimension of the cytoskeleton. Other cells were exposed to cytochalasin D, a fungal metabolite, which interferes with polymerization of actin from its globular to its filamentous form, and similarly were analyzed with respect to fractal dimension, viability (neutral red assay), and manner of death (annexin V fluorescence-activated cell scanning analysis). RESULTS: Induction of the inflammatory or heat-shock responses caused subtle and distinct rearrangement of the actin cytoskeleton. When these stimuli were applied in sequence, a synergistic interaction led to profound cytoskeletal collapse. Reversal of the sequence did not induce the cytoskeletal disruption. Cytochalasin D alone induced a dose-dependent cytoskeletal collapse indistinguishable from that caused by the acute phase-heat shock sequence that caused cell death by apoptosis. The effect of lower doses of Cytochalasin D could be potentiated by subsequent induction of the heat-shock response. CONCLUSIONS: Sequential stresses that mimic pathophysiologic "two-hit" stimuli induce a characteristic fractal rearrangement of the actin cytoskeleton. Because cytochalasin D-induced rearrangement of this cytoskeleton produced apoptosis indistinguishable from the stress-induced apoptosis, we conclude that the cytoskeletal rearrangement is likely a critical event in the pathway to apoptosis. This disruption of intracellular interconnections mirrors endotoxin-induced disruption in signals among organs and supports the mechanistic hypothesis that multiorgan dysfunction syndrome generally reflects disruption of signals and connections at several levels of biologic organization.

Actins↗
PubMedDate not supplied

Effect of PSC 833 on the efficacy of doxorubicin in vitro in a medullary thyroid carcinoma cell line.

In medullary carcinoma of the thyroid (MTC), multidrug resistance (MDR) remains the major obstacle to effective chemotherapy. In this work MDR was investigated in TT cells, a human MTC cell line. We studied the effect of an efficient MDR agent (SDZ PSC 833) on doxorubicin (DOX)-induced cytotoxicity in TT cells cultured in monolayers. The toxicity was evaluated with four tests: MTT test, lacticode-hydrogenase and glutathione assays, and neutral red uptake. PSC 833 (3 microM) partially reversed the resistance to DOX in vitro after a 48-hour coincubation, followed by a 24 hour-post incubation. Under these conditions, PSC 833 was not toxic at the concentration used. Our results suggest that PSC 833 has the potential to reverse the MDR phenotype in MTC cells.

Antibiotics, Antineoplastic↗
PubMed1998

Absorption enhancement, structural changes in tight junctions and cytotoxicity caused by palmitoyl carnitine in Caco-2 and IEC-18 cells.

Palmitoyl carnitine chloride (PCC) has been shown to be an effective enhancer of intestinal transport of hydrophilic molecules. The exact mechanism by which the epithelial barrier function is decreased is not clear. In an attempt to elucidate the mechanism of action of PCC, we studied the relationship among absorption enhancement, cell viability and tight junction protein localization in the human colonic Caco-2 cell line and the rat small intestinal cell line IEC-18. Filter-grown cells were exposed to 0 to 1 mM PCC for 30 min, and the efficacy of PCC treatment was determined by assessing the transepithelial electrical resistance and the apparent permeability for mannitol and PEG-4000. Membrane lysis and cytotoxicity were assessed by measurement of lactate dehydrogenase leakage and uptake of propidium iodide and neutral red. The immunolocalization of the tight junctional protein ZO-1 was quantified using CSLM and image-processing software. In both cell lines, PCC caused a dose-dependent decrease in transepithelial electrical resistance and a concomitant increase in the permeability for mannitol and PEG-4000. The transport enhancement was accompanied by an increase in apical membrane permeability and a reduction in cell viability. At higher PCC concentrations (>/=0.4 mM), the distribution of the tight junctional protein ZO-1 was changed and cells were unable to recover viability. PCC is effective as an absorption enhancer for hydrophilic macromolecules. However, lytic effects on the cell membrane and reduced cell viability were concomitant with transport enhancement.

Animals↗
PubMedDate not supplied

Arsenic(III), but not antimony(III), induces DNA-protein crosslinks.

Antimony compounds are supposed to resemble to arsenicals in some toxicological features. Comparative investigations with antimony and arsenic were performed to collect data on the genotoxicity of antimony, on which the knowledge is scarce. In comparison to trivalent arsenic, trivalent antimony proved to be five times less cytotoxic in the neutral red assay and one order of magnitude less genotoxic in the cytokinesis-block micronucleus test using V79 Chinese hamster cells. The data obtained in the comet assay showed that the As(III)-mediated generation of DNA-protein crosslinks and DNA strand lesions seem to be two independent processes. In contrast, Sb(III) induced DNA strand lesions but not DNA-protein crosslinks. Further studies will have to investigate whether the genotoxicity of Sb(III) is mediated by an inhibition of DNA repair as it seems to be the case for As(III).

Animals↗
PubMed1996

Age-related changes in neutral lipid content of Paramecium primaurelia as revealed by nile red.

Cellular neutral lipid content of Paramecium primaurelia was measured during culture and clonal life using nile red (9-diethylamino-5H-benzo[alpha] phenoxazine-5-one), a dye utilized for lipid analysis in both mammalian cells and in ciliated protozoa. Lipid droplets in P. primaurelia are concentrated in the anterior pole of the cell; their number is maximum in early log phase cells and decreases in late log phase cells. The quantitative determination of neutral lipids was obtained measuring the fluorescence from the excitation and emission spectra of 480 nm and 540 nm, respectively. Neutral lipid content decreases linearly during the log phase of growth while the decline is minimum during the stationary phase. In the late log phase, the amount is 30% of that of the early log-phase cells. Though the cell size declines too, cell area and lipid content decreases are not correlated in the middle log phase, because the maximum lipid reduction is obtained when the cell size is relatively constant. The cellular lipid content also changes during the clonal life. Neutral lipids decrease discontinuously (r = -0.75, P < 0.05) as the fission age increases. No relationship was found between lipid content and food vacuole formation during both culture and clonal life.

Animals↗